Language：English   Publishing type：Research paper (scientific journal)  

Click chemistry offers various applications through efficient bioorthogonal reactions. In bioimaging, pretargeting strategies have often been used, using click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution three-dimensional fluorescence imaging. Nevertheless, these techniques have faced a challenge in limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thoroughly staining whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed assay. The Click3D protocol exhibits a greater staining depth compared to conventional methods. Using Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. We have also accomplished whole-brain imaging of hypoxia by using the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier.

DOI： 10.1126/sciadv.ado8471

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here we show that gamma-glutamyltransferase 1 (GGT1)-SNP rs5751901 is an eQTL in pancreatic cells associated with PEP and a positive regulator of the IL-6 amplifier. More PEP patients had the GGT1 SNP rs5751901 risk allele (C) than that of non-PEP patients at Hokkaido University Hospital. Additionally, GGT1 expression and IL-6 amplifier activation were increased in PEP pancreas samples with the risk allele. A mechanistic analysis showed that IL-6-mediated STAT3 nuclear translocation and STAT3 phosphorylation were suppressed in GGT1-deficient cells. Furthermore, GGT1 directly associated with gp130, the signal-transducer of IL-6. Importantly, GGT1-deficiency suppressed inflammation development in a STAT3/NF-κB-dependent disease model. Thus, the risk allele of GGT1-SNP rs5751901 is involved in the pathogenesis of PEP via IL-6 amplifier activation. Therefore, the GGT1-STAT3 axis in pancreas may be a prognosis marker and therapeutic target for PEP.

DOI： 10.1038/s41598-024-60312-2

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbrc.2024.149666

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsnano.3c12716

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants including the next pandemic.

DOI： 10.1039/d3lc00572k

Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

DOI： 10.1093/intimm/dxad016

Language：English   Publishing type：Research paper (scientific journal)  

Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal-Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory-Tex), expressing both inhibitory receptors and effector molecules, into terminal-Tex, and inhibited tolerance induction. Adoptive transfer of transitory-Tex, but not terminal-Tex, into secondary recipients developed chronic GVHD. Transitory-Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory-Tex, not terminal-Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.

DOI： 10.1182/blood.2023019875

PubMed

Publishing type：Research paper (scientific journal)   Publisher：The American Association of Immunologists  

Abstract

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common β chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

DOI： 10.4049/jimmunol.2200567

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bio-Protocol, LLC  

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

DOI： 10.21769/BioProtoc.4644

Open Access

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn’s disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

DOI： 10.1093/intimm/dxad006

Other Link： https://academic.oup.com/intimm/article-pdf/35/7/313/50838496/dxad006.pdf

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cam4.5674

Open Access

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cam4.5674

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Dupuytren’s contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two single nucleotide polymorphisms (SNPs) associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and βTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.

DOI： 10.1093/intimm/dxad004

Other Link： https://academic.oup.com/intimm/article-pdf/35/7/303/50838446/dxad004.pdf

Language：English   Publishing type：Research paper (scientific journal)  

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.

DOI： 10.1038/s41467-022-32848-2

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.

DOI： 10.1111/cas.15034

Open Access

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor β and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.

DOI： 10.1111/cas.15007

Open Access

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.xpro.2020.100191

Open Access

Language：English   Publishing type：Part of collection (book)  

DOI： 10.1146/annurev-cellbio-111315-125001

Web of Science

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：日本癌学会  

Language：English   Publisher：(公社)日本生化学会  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese  

DOI： 10.15105/J01677.2015169593

CiNii Research

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Language：English   Presentation type：Oral presentation (invited, special)